MBNL1-dependent alternative splicing promotes neuronal differentiation through regulation of NUMA1 exon 16 during fibroblast-to-neuron reprogramming.

Introduction: Direct neuronal reprogramming enables the generation of neurons from somatic cells without passing through a pluripotent state, yet the post-transcriptional mechanisms that refine neuronal identity after fate induction remain poorly understood. Methods: We examined alternative splicing during fibroblast-to-neuron reprogramming and investigated the effects of MBNL1 knockdown on neuronal phenotype, transcriptomic and splicing changes, and NUMA1 exon 16 regulation. Results: MBNL1 knockdown establishes a distinct reprogramming state (AMmnp) characterized by enhanced neurite outgrowth and a more neuron-like differentiated phenotype, without significantly affecting conversion efficiency. Among MBNL1-dependent transcriptomic and splicing changes, NUMA1 exon 16 emerges as a key target, with exon inclusion reducing neuronal marker expression specifically in the AMmnp context, whereas exon skipping is associated with a more permissive neuronal phenotypic output. Discussion: Together, these findings position alternative splicing as an active regulatory layer that shapes neuronal identity and phenotypic output during reprogramming, linking MBNL1-dependent splicing control to cytoskeletal remodeling and neuronal differentiation.